Selection of transformed plants and plant cells in vitro often poses difficulties. Some of the technical challenges for selection include the clumpy nature of plant cell cultures; difficulty of single cells or protoplasts to grow and form clones; slow cell growth and polyploidy of the cells. Despite these problems a large number of successful selection experiments have been carried out to produce genetically engineered plants and cells for improving crop plants.
There are several types of in vitro selection techniques that can be used to obtain cells containing a trait of interest. These include selection for growth, selection for valuable compound production, auxotroph selection and resistance selection. Selection for resistance to an agent is generally the preferred kind of selection to screen for transformed cells or plants. Some of the examples include selection agents such as antibiotics and herbicides.
Selection for amino acid analog resistance in plants has been pursued for a number of years. A primary focus of this research has been directed to the enzyme anthranilate synthase (AS). AS catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimate) pathway toward the biosynthesis of tryptophan (Trp). As a branch-point enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic secondary compound biosynthesis.
In microbes, AS usually consists of two non-identical subunits, referred to as the alpha subunit (component I) and the beta subunit (component II). Component I can convert chorismate to anthranilate in the presence of high levels of ammonia (ammonia-dependent AS activity), whereas component II is responsible for the use of glutamine (hereinafter referred to as “Gln”) as the amino donor.
To investigate regulation of the Trp pathway, toxic analogs of Trp have been used in metabolic studies of plant cell cultures and as a tool to select mutants. Many of these studies have been conducted with the growth inhibitor 5-methyltryptophan (5MT). In addition, 5-methylanthranilate was used to isolate plant auxotrophic mutants defective in three different genes, trp1, trp2, and trp3. 5MT has also been used to identify mutants of Chlamydomonas reinhardtii. Mutants resistant to 5MT or alpha-methyltryptophan (αMT) were reported in Arabidopsis thaliana and other plants. The specificity of selection with these analogs has not been systematically investigated.
A feedback-insensitive AS gene (ASA1 mutant) has been identified by selection of mutagenized Arabidopsis seeds resistant to 6-methylanthranilate. A 5-methyltryptophan resistant Nicotiana tabacum anthranilate synthase gene (ASA2) was reported in U.S. Pat. No. 6,563,025 B1, the disclosure of which is hereby incorporated by reference.
One of the methods for developing transgenic plants is to transform plant cells in tissue culture with a plasmid or an expression cassette containing a promoter and selectable marker, which also contains a gene of interest. The gene of interest expresses the desired trait in the regenerated transgenic plant.
In plants, plastids generally differentiate into several forms based on the function they perform. For example, undifferentiated plastids (proplastids) may develop into amyloplasts (starch storage), chloroplasts (photosynthesis), etioplasts (chloroplasts that have not been exposed to light), elaioplasts (store fat), chromoplasts (pigment synthesis and storage), and leucoplasts (monoterpene synthesis). Each plastid contains multiple copies of the circular 75-250 kilo base plastid genome. The number of genome copies per plastid varies from more than 100 in rapidly dividing cells to about 20 or fewer in mature cells. The plastid genome contains about 100 genes encoding ribosomal and transfer ribonucleic acids (rRNAs and tRNAs) as well as proteins involved in photosynthesis and plastid gene transcription and translation.
Plastid transformation utilizes particle bombardment to deliver a transgene into the plastids where the integration is generally by homologous recombination using homologous (identical) plastid DNA sequences (generally about 1-4 kb long on each end) flanking the transgenes. The flanking sequences act as anchoring regions to initiate site-specific gene targeting in the plastid genome and homologous recombination during plastid transformation.
A plant-derived gene, such as an AS gene encoding an enzyme that is highly resistant to an amino acid analog or other agent, is an ideal selectable marker for the production of transgenic plants, because it minimizes the environmental concern regarding the use of non-plant resistance genes in plants for selection. Traditional selectable markers that are not of plant origin include nptII, which encodes kanamycin resistance.
Because many of the methods for inserting DNA into plants are inefficient and result in large numbers of plants of which only a tiny proportion are transformed, need exists for a robust plant-derived selection system. Selectable marker genes are therefore a pre-requisite for almost all of the current methods of plant transformation.